The present invention relates to pharmaceutical compositions comprising at least one sodium channel blocking compound in a suitable pharmaceutical vehicle.
The methods and compositions of the present invention provide a solution that meets the requirements for clinical use. The formulation can be administered systemically for indications including, but not limited to, pain requiring analgesia and treatment of drug dependence (See, U.S. Pat. No. 5,846,975). The formulation can be administered as described in A Method of Analgesia, Dong Q. et al, U.S. patent application Ser. No. 09/695,053, filed Oct. 25, 2000, Attorney Docket No. 3519-0103P.
Tetrodotoxin is a nonprotein neurotoxin with potent activity. It is found in diverse animal species, including puffer fish, goby fish, newt, frogs and the blue-ringed octopus.
As sodium channel blocking compounds with high selectivity, tetrodotoxin and saxitoxin specifically bind to a site on an extracellular region, either an SS1 region or an SS2 region, of a sodium channel alpha subunit. Surprisingly, compounds binding the SS1 or SS2 region of a sodium channel can produce long-acting and potent analgesia or anesthesia with no severe adverse effects (Dong Q. et al, supra, and Ku B. et al, U.S. patent application Ser. No. 09/702,826, filed Nov. 1, 2000, Attorney Docket No. 3519-0106P).
Tetrodotoxin (TTX) has a chemical formula of C11H17N3O8, and has a molecular weight of 319.28. The Merck Index, 10th Ed. (1983), states tetrodotoxin is the generic name for the compound octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-(1,3)dioxocino(6,5-d)-pyrimidine-4,7,10,11, 12-pentol, which has the following structure: 
The TTX molecule consists of a perhydroquinozoline group with a guanidine and six hydroxyls. Pure TTX is a white crystalline powder, odorless and tasteless. It turns black around 220xc2x0 C. without decomposition. TTX is soluble in acidic aqueous solution, but is not soluble in organic solvents. The pKa (aqueous) of TTX is 8.76. Thus TTX is a basic alkaloid. Aqueous solution of strong acids can decompose TTX, so usually TTX is dissolved in an aqueous solution of a weak organic acid. TTX is relatively thermally stable in neutral to weakly acidic solutions, but will be destroyed promptly in a strongly acidic or basic aqueous solution.
The present invention utilizes tetrodotoxin having a purity of at least 96% as the primary drug substance. Tetrodotoxin can be obtained by the technology described in A Method of Extracting Tetrodotoxin, Zhou M. et al, U.S. patent application Ser. No. 09/695,711, filed Oct. 25, 2000, Attorney Docket No. 3519-0101P. Further purification of TTX is described in Chinese patent application no.00132673.2, filed Nov. 22, 2000 and in U.S. patent application Ser. No. 09/818,863, filed Mar. 28, 2001, Attorney Docket No. 3519-0110P.
Analogs of tetrodotoxin can be used in the formulation of the invention. Preferred analogs of TTX are anhydrotetrodotoxin, tetrodaminotoxin, methoxytetrodotoxin, ethoxytetrodotoxin, deoxytetrodotoxin and tetrodonic acid.
Because TTX decomposes readily in gastric acid, oral ingestion is not considered to be a favored route of administration. Therefore, the compositions of the present invention are mainly for injection, preferably intramuscular injection. Most injectable formulations are solutions, and such properties as solubility, stability and safety of the drug must be considered when designing an injection (Bi D., Pharmaceutics, 4th Edition).
The calculation of the dose of TTX for injection is based upon the results of pre-clinical pharmacology and pharmacodynamics studies. The calculation of the clinical pharmaceutical dosage is based upon the dosage effective in animals. In general, it is calculated as ⅕ of the effective animal dosage. 50, 60, and 70 kg are used as human body weights, respectively.
The TTX analgesic ID50 (half inhibition dosage) in the acetic acid-induced twisting test in mice is 2.80 xcexcg/kg (intramuscularly, IM). Accordingly, the recommended clinical dosage for humans is:
2.80 xcexcg/kgxc3x97(⅕)xc3x9750 (60, 70)kg=28.0 (33.6, 39.2)xcexcg
The TTX effective dosage in the formalin-induced inflammation test in rats is 2.5 xcexcg/mg (IM) (P less than 0.01). Accordingly, the recommended clinical dosage for humans is:
2.50 xcexcg/kgxc3x97(⅕)xc3x9750 (60, 70)kg=25.0 (30.0, 35.0)xcexcg
It is also possible to calculate the initial clinical dosage based upon LD50 value. Considering the results of pharmacodynamics studies, the clinical dosage can be calculated as {fraction (1/50)} of the LD50. 50, 60, and 70 kg are used as human body weights, respectively. Our most recent study shows that the LD50 in mice is 11.1 xcexcg/kg (im), therefore, the suggested clinical dose is:
11.1 xcexcg/kgxc3x971/50xc3x9750(60,70)kg=11.1(13.32,15.54)xcexcg
Based upon the results of pharmacology studies and knowledge in the art, the concentration of TTX for injection is designed to be 15 xcexcg/mL, and the fill of a TTX injection is typically 1 or 2 mL.
TTX is a perhydroquinozoline compound that is only slightly soluble in water but soluble in an aqueous solution having an acidic or basic pH. Therefore, such commonly used auxiliary solvents as dilute acetic acid, hydrochloric acid and citric acid can be used dissolve TTX for formulation. Maintaining an acidic pH helps ensure the stability of a TTX formulation, as TTX is rather stable in weakly acidic solutions. The acid is preferably a weak organic acid, and formic acid, acetic acid or propionic acid is preferred.
It is preferable to include an adequate buffer, such as an acetate buffer, a citrate buffer, a phosphate buffer or a borate buffer, for the purpose of maintaining the pH of the formulation in an acceptable range. We have found (See Example 1) that the pH of a TTX formulation should be kept between 3.5-7.0, more preferably between 3.5-5.0. The buffer can be added in an amount of up to 0.5% by weight of the formulation. (A density of 1 g/ml is assumed for the formulated product.) As an example, a mixture of 55.5 ml of a 0.2M acetic acid solution and 4.5 ml of a 0.2M sodium acetate solution can be made, then added to a formulation in an amount of up to 5% by volume of the formulation. In this example, the buffer will constitute about 0.06% by weight of the formulation. Thus, the buffer can typically constitute about 0.1% by weight of the formulation.
In addition, a mixed solvent consisting of a glycol, preferably a C2-C6 alkane glycol, most preferably propylene glycol, and water is also desirable for improving the stability of TTX in a formulation. Addition of the glycol to the formulation results in acceptable stability with use of higher pH, providing a formulation that is more tolerated by the patient upon injection.
Environmental factors, such as temperature, light, humidity, and oxygen, were studied to determine their influence on the stability of TTX formulation so that a formulation and manufacturing technology that meet quality requirements can be devised. Test results (See Example 4) demonstrate that humidity and light have no significant effect on a TTX formulation, but increasing temperature will cause the content of TTX to decline. Therefore, a TTX formulation should be stored at low temperature so as to ensure its stability.
More components may be added to a TTX formulation in consideration of improving performance and convenience of storage, including viscosity increasing agents such as polyvinyl alcohol, celluloses, such as hydroxypropyl methyl cellulose and carbomer; preservatives, such as benzalkonium chloride, chlorobutanol, phenylmercuric acetate and phenyl mercuric nitrate; tonicity adjusters, such as sodium chloride, mannitol and glycerine; and penetration enhancers, such as glycols, oleic acid, alkyl amines and the like. A vasoconstrictor can also be added to the formulation. Combination formulations including the long-acting sodium channel blocking compound and an antibiotic, a steroidal or a non-steroidal anti-inflammatory drug and/or a vasoconstrictor are contemplated.
Regarding drug safety, examples described in A Method of Analgesia, Dong Q. et al, supra, have shown that TTX formulation complies with requirements for low toxicity, low hemolyzation and low local irritation. Dong Q. et al has also indicated that administration of a TTX formulation has no severe or non-reversible adverse effects.
A preferred composition of the present invention comprises substance of tetrodotoxin having a purity of 96% or higher as an active ingredient, an auxiliary solvent selected from dilute acetic acid, dilute hydrochloridic and citric acid, and a 5% acetic acid-sodium acetate buffer to maintain the pH between 3.5-5.0. High temperature should be avoided during manufacture of the formulation, and sterile processing is preferred. Excessive exposure to direct sunlight should be avoided during transportation, and it is recommended that the finished product be stored in a cool place, preferably at 4-20xc2x0 C.
Saxitoxin has physical and chemical properties similar to tetrodotoxin. Saxitoxin is found in dinoflagellates, which include Alexandrium tamarense, Gymnodinium catenatum, and Pyrodinium bahamense. Saxitoxin has the following structure: 
Saxitoxin is also stable in acidic solutions. Saxitoxin is a highly selective sodium channel blocker and produces analgesia or anesthesia by this mechanism. Therefore, saxitoxin can be used in the same manner as TTX in the compositions of the present invention.
The formulation of the present invention can optionally contain an auxiliary solvent selected from dilute acetic acid, dilute hydrochloric acid or citric acid, and a buffer selected from acetate buffers, citrate buffers, phosphate buffers and borate buffers.